Dengue serotype 1 attenuated strain

ABSTRACT

The invention relates to live attenuated VDV1 (VERO-Derived Dengue serotype 1 virus) strains which have been derived from the wild-type dengue-1 strain 16007 by passaging on PDK and sanitization on Vero cells and nucleic acids thereof. The invention further relates to a vaccine composition which comprises a VDV1 strain.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/633,411, filed Dec. 8, 2009, which is a divisional of U.S.application Ser. No. 11/449,876, filed Jun. 9, 2006, which claims thebenefit of U.S. provisional application 60/691,243, filed on Jun. 17,2005, all of said applications incorporated herein by reference.

The invention relates to new live attenuated VDV1 (VERO-Derived Dengueserotype 1 virus) strains which are derived from the wild-type dengue-1strain 16007 by passaging on PDK and Vero cells, and sanitization. Theinvention further relates to a vaccine composition which comprises suchVDV1 strain.

Dengue diseases are caused by four closely related, but antigenicallydistinct, virus serologic types (Gubler, 1988; Kautner et al., 1997;Rigau-Pérez et al., 1998; Vaughn et al., 1997), of the genus Flavivirus(Gubler, 1988). Infection with a dengue virus serotype can produce aspectrum of clinical illnesses ranging from a non-specific viralsyndrome to severe, fatal haemorrhagic disease. The incubation period ofdengue fever (DF) after the mosquito bite averages 4 days (range 3-14days). DF is characterised by biphasic fever, headache, pain in variousparts of the body, prostration, rash, lymphadenopathy and leukopenia(Kautner et al., 1997; Rigau-Pérez et al., 1998). The viremic period isthe same as of febrile illness (Vaughn et al., 1997). Recovery from DFis usually complete in 7 to 10 days but prolonged asthenia is common.Leukocytes and platelets counts decreases are frequent.

Dengue haemorrhagic fever (DHF) is a severe febrile diseasecharacterised by abnormalities of homeostasis and increased vascularpermeability that can lead to hypovolemia and hypotension (dengue shocksyndrome, DSS) often complicated by severe internal bleeding. The casefatality rate of DHF can be as high as 10% without therapy, but below 1%in most centres with therapeutic experience (WHO Technical Guide, 1986).

Routine laboratory diagnosis of dengue infections are based on virusisolation and/or the detection of dengue virus-specific antibodies.

Dengue disease is the second most important tropical infectious diseaseafter malaria, with over half of the world's population (2.5 billion)living in areas at risk for epidemic transmission. An estimated 50 to100 million cases of dengue, 500,000 hospitalised DHF patients and25,000 deaths occur each year. Dengue is endemic in Asia, the Pacific,Africa, Latin America, and the Caribbean. More than 100 tropicalcountries have endemic dengue virus infections, and DHF have beendocumented in more than 60 of these (Gubler, 2002; Monath, 1994). Anumber of well described factors appear to be involved in dengueinfections: population growth, unplanned and uncontrolled urbanisationparticularly in association with poverty, increased air travel, lack ofeffective mosquito control, and the deterioration of sanitary and publichealth infrastructure (Gubler, 2002). The awareness of dengue intravellers and expatriates is increasing (Shirtcliffe et al., 1998).Dengue has proven to be a major cause of febrile illness among US troopsduring deployments in dengue-endemic tropical areas (DeFraites et al.,1994).

The viruses are maintained in a cycle that involves humans and Aedesaegypti, a domestic, day-biting mosquito that prefers to feed on humans.Human infection is initiated by the injection of virus during bloodfeeding by an infected Aedes aegypti mosquito. Salivary virus isdeposited mainly in the extravascular tissues. The primary cell subsetinfected after inoculation is dendritic cells, which subsequentlymigrate to draining lymph nodes (Wu et al., 2000). After initialreplication in the skin and draining lymph nodes, virus appears in theblood during the acute febrile phase, generally for 3 to 5 days.

Monocytes and macrophages are with dendritic cells among the primarytarget of dengue virus. Protection against homotypic reinfection iscomplete and probably lifelong, but cross-protection between denguetypes lasts less than 12 weeks (Sabin, 1952). Consequently a subject canexperience a second infection with a different serotype. A second dengueinfection is a theoretical risk factor of developing severe denguedisease. However, DHF is multifactorial including: the strain of thevirus involved, as well as the age, immune status, and geneticpredisposition of the patient. Two factors play a major role in theoccurrence of DHF: a rapid viral replication with high viremia (theseverity of the disease being related to the level of viremia (Vaughn etal., 2000) and an important inflammatory response with release of highlevels of inflammatory mediators (Rothman and Ennis, 1999).

There is no specific treatment against Dengue diseases. The managementof DF is supportive with bed rest, control of fever and pain withantipyretics and analgesics, and adequate fluid intake. The treatment ofDHF needs correction of fluid loss, replacement of coagulation factors,and infusion of heparin.

Preventive measures presently rely on vector control and personalprotection measures, which are difficult to enforce and expensive. Novaccine against dengue is currently registered. Since the 4 serotypes ofdengue are circulating worldwide and since they are reported to beinvolved in cases of DHF, vaccination should ideally confer protectionagainst all 4 dengue virus serotypes.

Live attenuated vaccines (LAVs), which reproduce natural immunity, havebeen used for the development of vaccines against many diseases,including some viruses belonging to the same genus as dengue (examplesof commercially available flavivirus live-attenuated vaccines includeyellow fever and Japanese encephalitis vaccines). The advantages oflive-attenuated virus vaccines are their capacity of replication andinduction of both humoral and cellular immune responses. In addition,the immune response induced by a whole virion vaccine against thedifferent components of the virus (structural and non-structuralproteins) reproduced those induced by natural infection.

A dengue vaccine project was initiated in Thailand at the Centre forVaccine Development, Institute of Sciences and Technology forDevelopment Mahidol University. Candidate live-attenuated vaccines weresuccessfully developed, at a laboratory scale, for dengue serotype 1(strain 16007, passage 13=LAV1), serotype 2 (strain 16681, passage 53),and serotype 4 (strain 1036, passage 48) viruses in Primary Dog Kidney(PDK) Cells, and for serotype 3 (strain 16562) in Primary Green MonkeyKidney (PGMK) cells (passage 30) and Fetal Rhesus Lung (FRhL) cells(passage 3). These vaccines have been tested as monovalent (singleserotype), bivalent (two serotypes), trivalent (three serotypes), andtetravalent (all four serotypes) vaccines in Thai volunteers. Thosevaccines were found to be safe and immunogenic in children and in adults(Gubler, 1997). These LAV 1-4 strains have been described in EP 1159968in the name of the Mahidol University and were deposited before the CNCM(CNCM I-2480; CNCM I-2481; CNCM I-2482 and CNCM I-2483 respectively).

The complete sequence of the Dengue 1 Live-Attenuated Virus strain(LAV1) was established by R. Kinney et al. (CDC, Fort Collins). Sequencedifferences between parent DEN-1 strain 16007 (SEQ ID No.2) and LAV1(SEQ ID No.3) strain are described in Table 1. Thus, genetic comparisonof the wild-type virus strain 16007 and LAV1 strain showed a set of 14point mutations which could be linked to LAV1 attenuation.

TABLE 1 DEN-1 16007 and DEN-1 16007/PDK13  (LAV1) Sequence DifferencesLAV1 (DEN-1 Coordinates 16007/PDK13) 16007 Gene-aa position Nt aa nt aaE-130 Nt-1323 C Ala T Val E-203 Nt-1541 A Lys G Glu Nt-1543 G A E-204Nt-1545 A Lys G Arg E-211 Nt-1567 G Gln A Gln E-225 Nt-1608 T Leu C SerE-477 Nt-2363 G Val A Met NS1-92 Nt-2695 C Asp T Asp NS1-121 Nt-2782 TAla C Ala NS3-182 Nt-5063 A Lys G Glu NS3-510 Nt-6048 T Phe A TyrNS4A-144 Nt-6806 G Val A Met NS4B-168 Nt-7330 G Gln A Gln NS5-624Nt-9445 T Ser C Ser Nucleotide changes modifying the corresponding codonare indicated in bold.

The LAV1 strain which was initially established in 1983 was furtherrapidly identified as potential vaccine candidate (Bhamarapravati andYoksan, 1997).

However, at that time, transmission to humans of Spongiform Encephalitisthrough mammalian cultures was not perceived as a risk and the virus wasroutinely maintained in Primary Dog Kidney cells (PDK). Furthermore,this LAV1 strain corresponds to a heterogeneous population. Thisheterogeneity represents an additional risk due to a potential in vitroor in vivo selection of one of the strain present in the composition.

In view of these increasing concerns, the Applicant decided to set up asanitization process in order to get rid of any such risks. By firsttransferring the LAV1 vaccine strain from PDK to VERO cells and thentransfecting Vero cells with the purified genomic RNA of LAV1, followedby two successive steps of virus plaque purification the Applicantproduced a new Vero-Derived serotype 1 virus (VDV1).

This new VDV1 strain which has been thus derived by transfer to VEROcells and biological cloning differs from the LAV1 strain by sequence,an homogenous plaque size and temperature sensitivity but importantlyhas conserved some phenotypic and genotypic features of the LAV1 such ase.g. attenuation spots, small plaque phenotype, growth restriction athigh temperature, and has conserved the immunogenic features of the LAV1strains. These features make this new strain a valuable vaccinecandidate for prophylactic immunization in humans.

Definitions

“Dengue viruses” are positive-sense, single-stranded RNA virusesbelonging to the Flavivirus genus of the flaviridae family. In the caseof dengue serotype 1 (DEN-1) strain 16007, the entire sequence is 10735nucleotides long (SEQ ID No.2). The RNA genome contains a type I cap atthe 5′-end but lacks a 3′-end poly (A)-tail. The gene organization is5′-noncoding region (NCR), structural protein (capsid (C),premembrane/membrane (prM/M), envelope (E)) and non structural protein(NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and 3′ NCR. The viral RNA genome isassociated with the C proteins to form nucleocapsid (icosahedralsymmetry). As with other flaviviruses, the DEN viral genome encodes theuninterrupted open reading frame (ORF) which is translated to a singlepolyprotein.

Serial passaging of a virulent (disease-causing) strain of dengue-1results in the isolation of modified virus which are “live attenuated”,i.e., infectious, yet not capable of causing disease. These modifiedviruses are usually tested in monkeys to evaluate their attenuation.However, Humans are the only primates that exhibit signs of clinicaldisease. The viruses that cause mild (i.e. acceptable in terms ofregulatory purposes as presenting a positive benefit/risk ratio) to lowor no secondary effects (i.e. systemic events and/or biologicalabnormalities and/or local reactions) in the majority of the testedhumans but still infect and induce an immune response are called “liveattenuated”.

The term “LAV” denotes live attenuated Dengue viral strains. In thecontext of the invention “LAVs” are live attenuated strains initiallyderived from the Dengue serotype 1 (DEN-1) strain 16007 by passages,e.g. 10, 11, 12 or 13 passages, in Primary Dog Kidney (PDK) Cells. Forinstance “LAV1/PDK13” is the attenuated strain established after 13passages of strain 16007 in PDK cells (also named DEN-1 16007/PDK13).LAV1/PDK13 nucleotide sequence is shown in SEQ ID No.3.

“VDV1” is meant a LAV obtainable by the sanitization process disclosedin the present application. A VDV1 is thus a biological clone(homogeneous) VERO- adapted Dengue serotype 1 virus capable of inducinga specific humoral immune response including neutralizing antibodies inprimate especially in humans. The VDV1 strains of the invention can beeasily reconstructed starting directly from the here disclosed VDV1sequences. The induction of a specific humoral immune response can beeasily determined by an ELISA assay. The presence of neutralisingantibody in the serum of a vaccinee is evaluated by the plaque reductionneutralization test as described in section 4.1.2.2. A serum isconsidered to be positive for the presence of neutralizing antibodieswhen the neutralizing antibody titer thus determined is at leastsuperior or equal to 1:10.

The terms “mutation” means any detectable change in genetic material,e.g. DNA, RNA, cDNA, or any process, mechanism, or result of such achange. Mutations include substitutions of one or more nucleotides. Inthe context of the instant application, mutations identified in dengue-1virus genomic sequence or polyprotein are designated pursuant to thenomenclature of Dunnen and Antonarakis (2000). As defined by Dunnen andAntonarakis at the nucleic acid level, substitutions are designated by“>”, e.g. “31A>G” denotes that at nucleotide 31 of the referencesequence a A is changed to a G.

Variations at the protein level describe the consequence of the mutationand are reported as follows. Stop codons are designated by X (e.g. R97Xdenotes a change of Arg96 to a termination codon). Amino acidsubstitutions are designated for instant by “S9G”, which means that Serin position 9 is replaced by Gly.

VERO-Derived Dengue Serotype 1 Viruses (VDV1)

The composition of the previously developed dengue-1 vaccine candidateLAV1 was improved by a sanitization process.

The VERO-Derived Dengue serotype 1 viruses (VDV1) disclosed herein usethe DEN-1 16007 virus attenuated by serial passages on PDK cells. VDV1contains the whole genomic sequence of the live-attenuated DEN-1 virus,and bears the same spots which have been linked to attenuation as theoriginal LAV1 strain that was tested in humans.

Sanitization of the LAV1 vaccine candidate was performed by removingproteins and introducing only purified viral genomic material into Verocells. More specifically, sanitization of the strain was performed in 2steps:

1) Amplification of DEN16007/PDK11 (LAV1/PDK11) on Vero cells, at 32° C.

2) Purification and transfection of viral RNA into Vero cells.

Step 1 has been carried out by one passage of LAV1/PDK11 on Vero cells.For that purpose, Vero cells were infected with LAV1/PDK11 at a moi of0.01 and incubated at 32° C. for 5 days.

For step 2, advantage was taken of the fact that the viral genome is aninfectious

RNA, which means that it is able, when introduced into a cell, toreconstitute a complete infectious virus. The second purification andtransfection step thus comprised the steps consisting of:

a) extracting and purifying viral RNA from plaque-purified viruses;

b) advantageously associating of the purified RNA with cationic lipids;

c) transfecting Vero cell, in particular Vero cell LS10;

d) recovering of the neo-synthesized viruses; and

e) purifying a VDV strain by plaque purification and optionallyamplifying it in host cells, especially Vero cells.

The Vero cell technology is a well-known technology which has been usedfor different commercial products (injectable and oral polio vaccines,rabies vaccine). In the present invention qualified Vero cells wereadvantageously used to guarantee the absence of any risks potentiallylinked to the presence of adventitious agents. By “qualified VERO cells”is meant cells or cell lines for which culture conditions are known andis such that the said cells are free from any adventitious agents. Theseinclude e.g. the VERO cell LS10 of Sanofi Pasteur.

The thus isolated VDV strains are classically stored either in the formof a freezed composition or in the form of a lyophilised product. Forthat purpose, the VDV can be mixed with a diluent classically a bufferedaqueous solution comprising cryoprotective compounds such a sugaralcohol and stabilizer. The pH before freezing or lyophilisation isadvantageously settled in the range of 6 to 9, e.g. around 7 such as apH of 7.5+/−0.2 as determined by a pH meter at RT. Before use, thelyophilised product is mixed with a pharmaceutically diluent orexcipient such as a sterile NaCl 4% solution to reconstitute a liquidimmunogenic composition or vaccine.

Sequencing, at attenuation-specific loci, of virus recovered aftertransfection, did not reveal any mutation, compared to the LAV1/PDK13strain. The biologically cloned VDV1 virus exhibits a homogenous plaquephenotype and a remarkable genetic stability with regard to its LAV1parent as it can be deduced especially from the conservation of theattenuation genotype.

VDV1 strain was sequenced and compared with the serotype 1 Dengue LiveAttenuated Virus (LAV1/PDK13) strain sequence (SEQ ID No 3). A set of 3nucleotide differences was found with regard to the reference LAV1sequence. One of them is silent at the amino acid level (position 2719).The two others (positions 5962 and 7947) are located in non-structuralpeptides coding sequences (NS3-481 and NS5-125, respectively). None ofthese differences corresponds to any of the LAV1 attenuation positions.

The invention thus provides for live attenuated dengue-1 virus strainsthat have been obtained from the wild type virus DEN-1 16007 attenuatedby serial passages on PDK cells and then by passage and sanitization onVERO cells. In particular the attenuated strains of the inventioncomprise at least the identified sequence mutations (non-silent andoptionally silent) relative to the nucleotide sequence or polyproteinsequence of the wild-type DEN-1 16007 and LAV1/PDK13 strains.

Accordingly, the invention relates to an isolated live attenuateddengue-1 virus strain which comprises, or consists of, the sequence ofLAV1/PDK13 strain (SEQ ID No.3) wherein at least nucleotides atpositions 5962 and 7947, and optionally 2719, are mutated, with theproviso that the following nucleotides are not mutated: 1323, 1541,1543, 1545, 1567, 1608, 2363, 2695, 2782, 5063, 6048, 6806, 7330, and9445. Preferably, the mutations are substitutions. Preferably, thenucleotide at position 5962 is A, the nucleotide at position 7947 is G.Still preferably, the isolated strain according to the inventioncontains sequence SEQ ID No.3 which comprises the mutations 2719 G>A,5962 C>A, and 7947 A>G.

Hence, a live attenuated dengue-1 virus strain according to theinvention comprises the sequence of wild-type dengue-1 strain 16007 (SEQID No.2) wherein said sequence comprises at least the mutations 1323T>C, 1541 G>A, 1543 A>G, 1545 G>A, 1567 A>G, 1608 C>T, 2363 A>G, 2695T>C, 2782 C>T, 5063 G>A , 5962 C>A, 6048 A>T, 6806 A>G, 7330 A>G, 7947A>G, and 9445 C>T. Preferably, a live attenuated strain according to theinvention further comprises the mutation 2719 G>A by reference to thenucleotide sequence of wild-type strain 16007 (SEQ ID No.2).

The live attenuated dengue-1 virus strains according to the inventionencompass variant strains that comprise a sequence SEQ ID No.3 mutatedin positions 5962 and 7947, as defined above, and that further comprisea substitution of one or more nucleotides in a given codon position thatresults in no alteration in the amino acid encoded at that position.

Advantageously, the live attenuated dengue-1 virus strain according tothe invention comprises a sequence which differs by a limited number ofmutations, e.g. no more than 5, still preferably no more than 2, fromSEQ ID No.1.

Preferably, the genomic sequence of a dengue-1 virus strain according tothe invention consists of the nucleotide sequence SEQ ID No.1.

The invention also relates to live attenuated dengue-1 strains that maybe derived from the VDV1 strain of sequence SEQ ID No.1 by furtherpassages on cells, in particular Vero cells.

The invention also relates to an isolated nucleic acid which comprises,or consists of, the DNA sequence SEQ ID No.1 or its equivalent RNAsequence.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix.

As used herein, by RNA sequence “equivalent” to SEQ ID No.1 is meant asequence SEQ ID No.1 wherein deoxythymidines have been replaced byuridines. As SEQ ID No.1 constitutes VDV1 cDNA sequence, the equivalentRNA sequence thus corresponds to the positive strand RNA of VDV1.

The invention further relates to the polyprotein of sequence SEQ IDNo.41 and to fragments thereof. SEQ ID No.41 is the sequence of thepolyprotein encoded by SEQ ID No.1

A “fragment” of a reference protein is meant a polypeptide whichsequence comprises a chain of consecutive amino acids of the referenceprotein. A fragment may be at least 8, at least 12, at least 20, aminoacid long.

Said fragments of the polyprotein of sequence SEQ ID No.41 comprise atleast a lysine at position 481 of NS3 protein (position 1956 of SEQ IDNo.41), and/or an arginine at position 125 of NS5 protein (position 2618of SEQ ID No.41).

According to an embodiment the fragment of the polyprotein encoded bySEQ ID No.1 is or comprises NS3 protein and/or NS5 protein.

Immunogenic and Vaccine Compositions

The invention also relates to an immunogenic composition, suitable to beused as a vaccine, which comprises a VDV1 strain according to theinvention.

The immunogenic compositions according to the invention elicit aspecific humoral immune response toward the dengue virus comprisingneutralizing antibodies.

Preferably, the immunogenic composition is a vaccine.

According to an embodiment, the immunogenic is a monovalent composition,i.e. it elicits a specific immune response and/or confers protectionagainst Dengue-1 virus only.

According to another embodiment, the invention relates to a multivalentdengue immunogenic composition. Such a multivalent immunogeniccomposition or vaccine may be obtained by combining individualmonovalent dengue vaccines. The immunogenic or vaccine composition mayfurther comprise at least a live attenuated dengue virus of anotherserotype. In particular, the immunogenic or vaccine composition maycomprise a VDV1 according to the invention in combination with at leasta live attenuated dengue virus selected from the group consisting ofserotype 2, serotype 3, and serotype 4.

Preferably, the immunogenic or vaccine composition may be a tetravalentdengue vaccine composition, i.e. a vaccine composition that comprises aVDV1 according to the invention in combination with a live attenuateddengue-2 virus strain, a live attenuated dengue-3 virus strain and alive attenuated dengue-4 virus strain.

Live attenuated dengue-2, dengue-3 and dengue-4 virus strains have beendescribed previously. Reference may be made to the live-attenuatedvaccines that were developed by Mahidol University by passaging dengueserotype 2 (strain 16681, passage 53; LAV2), and serotype 4 (strain1036, passage 48, LAV4) viruses in Primary Dog Kidney (PDK) Cells, andfor serotype 3 (strain 16562) in Primary Green Monkey Kidney (PGMK)cells (passage 30) and Fetal Rhesus Lung (FRhL) cells (passage 3)(LAV3). The nucleotide sequences of LAV2 (SEQ ID No.42), LAV3 (SEQ IDNo.43), and LAV4 (SEQ ID No.44) are shown in the annexed sequencelisting.

Advantageously, a live attenuated dengue-2 strain may correspond to aVDV2 strain which has been obtained from the LAV2 strain developed byMahidol by a process of sanitization on Vero cells. In particular a liveattenuated dengue-2 strain (VDV2) may comprise, and advantageouslyconsists of the sequence SEQ ID No.40.

Immunogenic compositions including vaccines may be prepared asinjectables which can correspond to liquid solutions, suspensions oremulsions. The active immunogenic ingredients may be mixed withpharmaceutically acceptable excipients which are compatible therewith.

The immunogenic compositions or vaccines according to the presentinvention may be prepared using any conventional method known to thoseskilled in the art. Conventionally the antigens according to theinvention are mixed with a pharmaceutically acceptable diluent orexcipient, such as water or phosphate buffered saline solution, wettingagents, fillers, emulsifier stabilizer. The excipient or diluent will beselected as a function of the pharmaceutical form chosen, of the methodand route of administration and also of pharmaceutical practice.Suitable excipients or diluents and also the requirements in terms ofpharmaceutical formulation, are described in Remington's PharmaceuticalSciences, which represents a reference book in this field.

Preferably, the immunogenic composition or vaccine corresponds to aninjectable composition comprising an aqueous buffered solution tomaintain e.g. a pH (as determined at RT with a pH meter) in the range of6 to 9.

The composition according to the invention may further comprise anadjuvant, i.e. a substance which improves, or enhances, the immuneresponse elicited by the VDV1 strain. Any pharmaceutically acceptableadjuvant or mixture of adjuvants conventionally used in the field ofhuman vaccines may be used for this purpose.

The immunogenic compositions or vaccines according to the invention maybe administered by any conventional route usually used in the field ofhuman vaccines, such as the parenteral (e.g. intradermal, subcutaneous,intramuscular) route In the context of the present invention immunogeniccompositions or vaccines are preferably injectable compositionsadministered subcutaneously in the deltoid region.

Method for Immunizing

The invention further provides for a method of immunizing a host in needthereof against a dengue infection which comprises administering thehost with an immunoeffective amount of an immunogenic composition or avaccine according to the invention.

A “host in need thereof” denotes a person at risk for dengue infection,i.e. individuals travelling to regions where dengue virus infection ispresent, and also inhabitants of those regions.

The route of administration is any conventional route used in thevaccine field. The choice of administration route depends on theformulation that is selected. Preferably, the immunogenic composition orvaccine corresponds to an injectable composition administered viasubcutaneous route, advantageously in the deltoid region.

The amount of LAV or VDV in particular VDV1 in the immunogeniccompositions or vaccines may be conveniently expressed in viral plaqueforming unit (PFU) unit or Cell Culture Infectious Dose 50% (CCID₅₀)dosage form and prepared by using conventional pharmaceuticaltechniques. For instance, the composition according to the invention maybe prepared in dosage form containing 10 to 10⁶ CCID₅₀, or from 10³ to10⁵ CCID₅₀ of LAV or VDV, for instance 4 ±0.5 log₁₀ CCID₅₀ of VDV1strain for a monovalent composition. Where the composition ismultivalent, to reduce the possibility of viral interference and thus toachieve a balanced immune response (i.e. an immune response against allthe serotype contained in the composition), the amounts of each of thedifferent dengue serotypes present in the administered vaccines may notbe equal.

An “immunoeffective amount” is an amount which is capable of inducing aspecific humoral immune response comprising neutralising antibodies inthe serum of a vaccinee, as evaluated by the plaque reductionneutralization test as described in section 4.1.2.2; a serum beingconsidered to be positive for the presence of neutralizing antibodieswhen the neutralizing antibody titer thus determined is at leastsuperior or equal to 1:10.

The volume of administration may vary depending on the route ofadministration. Subcutaneous injections may range in volume from about0.1 ml to 1.0 ml, preferably 0.5 ml.

The optimal time for administration of the composition is about one tothree months before the initial exposure to the dengue virus. Thevaccines of the invention can be administered as prophylactic agents inadults or children at risk of Dengue infection. The targeted populationthus encompasses persons which are naïve as well as well as non-naïvewith regards to dengue virus. The vaccines of the invention can beadministered in a single dose or, optionally, administration can involvethe use of a priming dose followed by a booster dose that isadministered, e.g. 2-6 months later, as determined to be appropriate bythose of skill in the art.

The invention will be further described in view of the following figuresand examples.

FIGURES

FIG. 1 is a summary of History of VDV1 pre-master seed.

FIG. 2 is a flow chart that summarises the developed manufacturingprocess that gives rise to the Filled Product (monovalent), “ready touse” doses.

FIG. 3 is a diagrammatic representation of VDV1 genome map. The abovearrow is the polyprotein coding sequence. The below arrows representmature peptides coding sequence. The vertical bars symbolize thenucleotidic variations between wild-type dengue 1 strain16007 and LAV1strain. The stars designate the nucleotidic variations between LAV1 andVDV1.

FIG. 4 shows plaque size analysis after 7 days of incubation at 37° C.for dengue-1 viruses LAV1, VDV1, and strain 16007.

FIG. 5 is a graphic analysis showing plaque size distribution fordengue-1 viruses LAV1, VDV1, and strain 16007.

FIG. 6 is a summary of Trial Design for assessment of safety of VDV1monovalent in healthy flavivirus-naïve adults.

EXAMPLES Example 1 Sanitization

1.1 Viral RNA Purification

It was initialy intended to perform sanitization of LAV1 by purifyingand transfecting viral RNA directly extracted from an early seed of thevaccine strain, DEN-16007/PDK10 or DEN-16007/PDK11 (produced by SanofiPasteur. Titer: 4.60 logTCID₅₀/ml). Eight unsuccessful assays werecarried out in that way, with RNA quantities varying from 10³ to 10⁷copies. It was then decided to perform one adaptation passage on Verocells, before RNA extraction and transfection.

Vero cells (VERO LS10 p142 to 145) were infected with a sample of themaster seed DEN-1/PDK11, at m.o.i 0.01, and incubated at 32° C. for 5days. Culture medium was then replaced by infection medium (containing10 mM MgSO₄). Clear cytopathic effects were visible the following day,and presence of viral RNA in culture supernatant was confirmed byRT-PCR. Culture medium was collected at day 8 post-infection, dilutedwith an equivalent volume of an aqueous buffered solution comprisingcryoprotective agents (pH=7.5) and kept frozen at −70° C. until use.This Vero-amplified virus was named DEN-1/V100. Its infectious titer wasdetermined on Vero cells and was of 6.9 logTCID₅₀/ml.

The RNA purification and transfection process was performed as follows.DEN-1 V100 suspension was diluted in order to contain at least 3×10⁴ andup to 3×10⁷ TCID₅₀ or PFU of virus per milliliter. One unit of benzonasediluted in 0.01 ml of

William's medium was added to 0.5 ml of virus, in order to digest DNA orRNA molecules from cellular origin, and the solution was incubated for 2hours at 4° C. on an agitator. At the end of incubation step, 0.65 ml ofa denaturing buffer containing guanidium chloride, detergent (SDS), andβmercaptoethanol (RTL-βmercaptoethanol buffer, provided in the kitRNeasy Mini kit, Qiagen Ref. 74104) were added and proteins wereextracted once with phenol/chloroform (1/1) vol/vol and once withchloroform vol/vol, followed by centrifugation for 5 min at 14,000 rpmat room temperature. After each extraction, the aqueous phase wascollected, taking care not to collect material (white precipitate) atthe interface, and transferred to a clean 1 ml-Eppendorf tube. The RNAsolution was then applied onto a QIAgen column following therecommendations of the manufacturer (RNeasy minikit, QIAgen), in orderto remove traces of solvent, and eluted with 0.06 ml of nuclease-freeH2O water. The presence of viral RNA was confirmed by quantitativeRT-PCR, using a reference curve established with known quantities ofvirus, in TCID₅₀/ml.

1.2 Transfection of Vero Cells with Purified RNA

Transfection was performed using lipofectamine (LF2000 Reagent, LifeTechnologies), a mixture of cationic lipids that associate to RNAthrough charge interactions and allows transfer of the complexes intothe cytoplasm of the cells by fusion with the cell membrane. The optimalquantity of LF2000 reagent was determined in a preliminary experiment byincubating Vero cells, plated 16 to 24 hours before (0.3-0.5×10⁶ cellsper well in a 6 wells plate) with increasing doses (5 to 20 μl) oflipofectamine. Cells were then incubating 4 to 5 hours at 32° C., 5%CO₂, before replacing the medium by fresh culture medium without FCS,and the incubation was continued overnight at 32° C. Toxicity (round,refringent or floating cells, homogeneity of the cell monolayer) waschecked regularly for 48 hours, under an inverted microscope. Thehighest dose of lipofectamine that was not toxic under these conditionswas 10 μl and was chosen for RNA transfection.

Four transfections were carried out in parallel, using 1/10 of thepurified RNA preparation (corresponding to about 4×10⁵ TCID₅₀). Twelvemicroliters of viral RNA solution were diluted in 500 μl of OptiMEMmedium (GIBCO) containing 10 μl of LF2000 Reagent (a mixture of cationiclipids that associate to RNA through charge interactions, and allowtransfer of the complexes into the cytoplasm of the cells by fusion withthe cell membrane). 200 ng of yeast tRNA were added as carrier in 2 outof the 4 reactions.

The 4 transfection mixes were allowed to precipitate for 10 min at roomtemperature before addition to 6-wells plates of confluent Vero cells.After 4 hours of incubation at 32° C., transfection mix was removed andcells were rinsed once in PBS. Three milliliters of post-transfectionmedium (Williams, GIBCO) were added, and incubation was continued for 5days at 32° C. Culture medium was then replaced by 3 ml of Dengueinfection medium (Williams supplemented with 10 mM MgSO₄).

Moderate toxic effects were observed 24 hours post-transfection anddisappeared on day 3. Typical cytopathic effects (round, refringentcells) were detected 6 to 8 days post-transfection in all transfectionassays. Release of virus in the supernatant of these cells was confirmedby qRT-PCR. Culture fluids (3 ml) were collected at day 6 and at day 8post-transfection, and pooled. The viruses were diluted with 6 ml of anaqueous buffered solution comprising cryoprotective agents (pH=7.5) andfrozen until further amplification.

The four viral solutions such obtained after transfection were named TV(for Transfection in Vero cells) 100, TV200, TV300 and TV400, andexhibited similar infectious titers (see below):

TV100: 6.95 log TCID₅₀

TV200: 6.80 log TCID₅₀

TV300: 6.80 log TCID₅₀

TV400: 6.85 log TCID₅₀

Of note, transfection efficiency was not significantly increased insamples transfected in presence of tRNA (TV300 and TV400).

1.3 Characterization of Viruses Recovered After Transfection

Plaques sizes of DEN1-V100 and TV100 to 400 were determined. Briefly,Vero cells were plated at a density of 1.000.000 cells/cm² in culturemedium containing 4 of FBS. After overnight incubation, the medium wasremoved and cells were infected with serial twofold or fivefolddilutions of virus. After 1.5 hour at 37° C. 5% CO₂, the inoculum wasremoved and cells were incubated at 37° C. 5% CO₂ in Mimimal EagleMedium (MEM) containing 1.26% methylcellulose and 10% FBS. After 11 daysof incubation, plates were fixed 20 minutes in cold acetone at −20° C.and revealed by immuno-coloration with a flavivirus-specific mAb,diluted at 2.5 μg/ml. Viral plaques were measured using an imageanalysis software (Saisam/Microvision)

As a control, DEN-1 16007 and LAV1 were plated in parallel. The data arepresented in Table 1.

TABLE 1 Plaques size of DEN-1 16007, LAV1, V100 (before transfection)and TVX00 (after transfection) Phenotype Large Small Step Virus PlaquePlaque DEN1 16007 (wt) 42 0 Master Seed/PDK11 MS-16007/PDK11 0 91Working Seed/PDK12 WS LST-22 0 84 Bulk Seed/PDK13 D1-0010R1 0 188 VEROamplification V100 0 90 Transfected viruses TV100 0 115 TV200 1 90 TV3000 92 TV400 0 107

The V100 virus, amplified on Vero cells, exhibits a homogeneous smallplaque (SP) phenotype. Plaques are slightly larger than observed in thedifferent samples of LAV1 (2-3 mm diameter instead of <2 mm). This SPphenotype is retained in the viruses recovered after transfection. Onelarge plaque (LP), among 90 virus plated, was detected in TV200 sample.However, this proportion shifted to 10 LP for 82 SP plated after justone amplification passage on VERO cells suggesting that the LPpopulation was dominant.

Of note, sequencing of attenuation-critical positions performed inparallel did not reveal any mutation in transfected viruses, compared toLAV1.

1.4 Plaque-Purifications

A sample of DEN-1/TV100 virus, presenting a homogenous SP phenotype waschosen for plaque-purification. Briefly, Vero cells were plated in6-well plates and infected with serial dilutions of virus, in order toget between 1 and 20 plaques by plate. After 1.5 hour at 37° C. 5% CO₂,the inoculum was removed and cells were incubated under 3 ml of solidmedium composed of MEM-10% FCS pre-heated at 42° C. and mixedextemporaneally with 2% of melted agarose equilibrated at 42° C. Themedium was allowed to solidify at room temperature for 30 min; underflow hood, and plates were incubated in inverted position for 10 days at32° C.-5% CO₂. A second layer of the same medium supplemented with 0.01%of neutral red was then added and plates were incubated for anadditional night at 32° C. Four well-isolated small plaques were pickedunder sterile conditions using a micro-pipet equipped with a 0.1 ml tip,and transferred into sterile tubes containing 0.2 ml of MEM-4% FCS. Thesuspension was homogeneized by vortexing, serially diluted in the samemedium, and immediately used to infect 6-well plates of Vero cells. Theprotocol was repeated and a second picking of six SP was done. Eachpicked plaque was diluted in 1 ml of medium, before amplification onVero cells, in T25 cm² flasks. Culture medium was collected at day 6post-infection, diluted with the same volume of an aqueous bufferedsolution comprising cryoprotective agent (pH 7.5) and frozen at −70° C.All these steps were performed at 32° C.

Plaque-purified viruses were named DEN-1/TV111, DEN-1/TV112,DEN-1/TV121, DEN-1/TV131, DNE-1/TV132 and DEN-1/TV141, respectively.Infectious titers were determined on Vero cells (see below):

TV111=6,85 LogCCID₅₀/ml TV112=6,80 LogCCID₅₀/ml

TV121=6,80 LogCCID₅₀/ml TV131=6,70 LogCCID₅₀/ml

TV132=6,45 LogCCID₅₀/ml TV141=5,70 LogCCID₅₀/ml

A second amplification on Vero cells was carried out for three clones:TV111, TV112 and TV121.

1.5 Characterization of Cloned Virus

Plaques size of DEN-1/TV111, DEN-1/TV112, DEN-1/TV121, DEN-1/TV131,DEN-1 TV132 and DEN-1/TV141 candidates were determined. Spot-sequencingof specific attenuation loci was also performed and revealed no mutation(Table 2).

TABLE 2 Sequencing at attenuation-specific spots of DEN-1 viruses E NS1NS3 NS4A NS4B NS5 Step/cell Virus 1323 1541 1543 1545 1567 1608 23632695 2782 5063 6048 6806 7330 9445 Non attenuated/PGMK DEN-1  T G A G AC A T C G A A A C 16007 Vaccine/PDK DEN-1  C A G A G T G C T A T G G T16007/ PDK-13 TV111 C A G A G T G C T A T G G T TV112 C A G A G T G C TA T G G T 2nd plaque- TV121 C A G A G T G C T A T G G Tpurification/VERO TV131 C A G A G T G C T A T G G T TV132 C A G A G T GC T A T G G T TV141 C A G A G T G C T A T G G T Pre-master VDV1  C A G AG T G C T A T G G T seed/VERO (VERO-6) Nucleotides position areindicated below each gene and referred of DEN-1 16007 strain SEQ ID No2.

In absence of any other criterion able to differentiate between theseclones, TV121 was arbitrarily chosen as pre-master for VDV1.

In conclusion, a total number of 6 passages on VERO cells were carriedout to adapt and clone the initial DEN-1 16007/PDK11 attenuated strain.Viral RNA was purified and transfected into qualified VERO cells, inconditions fitting with an industrial application (environmentalcontrol, traceability of raw material and experiments, certificate ofanalysis for animal-derived products). The VERO-adapted strain wascloned by plaque-purification to generate pre-master seed of VDV1vaccine candidate, at VERO passage number 6.

Contrary to LAV1, VDV1 presents a homogenous small plaque sizephenotype. Furthermore, no mutation was identified atattenuation-specific positions. Further characterizations have beenperformed then by determining bulk VDV1 complete sequence and phenotypictesting.

Example 2 Sequencing

The complete sequence of the virus was generated according to thefollowing strategy. Starting from a VDV1-containing sample, the genomicRNA was extracted and purified, retro-transcribed into cDNA. Then alloverlapping PCR amplifications were performed from the cDNA, withaddition of the sequencing tags at both ends of each PCR product. Allindividual sequences were generated in automated devices and analysed.Next step consisted of the genome reconstruction by multiple alignmentsof all individual sequences. At this point, each unexpected nucleotidechange, with regard to the reference sequence, was carefully re-analysedby going back to raw data. Such change was systematically confirmed byanother sequence performed from a new PCR product. Once all ambiguitieswere solved, the sequenced virus genome was completed, and the newmolecule was created in Vector NTi database. It can be used for intergenomes analysis, by multiple sequence alignment.

2.1 Materials

2.1.1 Viruses

The viruses to which it is referred here are DEN-1 16007; LAV-1/PDK13;VDV1, the sequences of which are given in the attached sequence listing.The complete genome sequence of these viruses is 10735 nucleotides inlength.

2.1.2 Primers

All primers have been designed in Seqweb bioinformatics package(Accelrys), primer design module (Table 3).

TABLE 3 list of RT-PCT and sequencing primers Primer RT-PCR NamePrimers sequences NtStart NtEnd length length Overlap D1 01+GTTTTCCCAGTCACGACtacgtggaccgacaagaacag    12    32 38 897 −32(SEQ ID No. 4) D1 01− AACAGCTATGACCATGggatggagttaccagcatcag   928   90837 (SEQ ID No. 5) D1 02+ GTTTTCCCAGTCACGACtgaacaccgacgagacaaac   688  707 37 201 (SEQ ID No. 6) D1 02− AACAGCTATGACCATGaggtccaaggcagtggtaag 1598  1579 36 892 (SEQ ID No. 7) D1 03+GTTTTCCCAGTCACGACttggaaatgagaccacagaac  1386  1406 38 173 (SEQ ID No. 8)D1 03− AACAGCTATGACCATGgaaacaccgctgaacaaaac  2289  2270 36 885(SEQ ID No. 9) D1 04+ GTTTTCCCAGTCACGACggttcaagaagggaagcag  2106  212436 146 (SEQ ID No. 10) D1 04− AACAGCTATGACCATGttctatccagtaccccatgtc 3028  3008 37 903 (SEQ ID No. 11) D1 05+GTTTTCCCAGTCACGACcagaataccaccttcatcatcg  2804  2825 39 183(SEQ ID No. 12) D1 05− AACAGCTATGACCATGttcccatccccatcttgtc  3689  367135 868 (SEQ ID No. 13) D1 06+ GTTTTCCCAGTCACGACggaaatcagaccagtcaaggag 3418  3439 39 232 (SEQ ID No. 14) D1 06−AACAGCTATGACCATGtgttgtgtgaggcaccagag  4349  4330 36 913 (SEQ ID No. 15)D1 07+ GTTTTCCCAGTCACGACgcaaaccactaaccatgtttc  4077  4097 38 233(SEQ ID No. 16) D1 07− AACAGCTATGACCATGccacttgttgtcaccactc  4995  497735 901 (SEQ ID No. 17) D1 08+ GTTTTCCCAGTCACGACccaagggaagagactggaac 4699  4718 37 259 (SEQ ID No. 18) D1 08−AACAGCTATGACCATGtcctgatttgatgcttggaac  5626  5606 37 908 (SEQ ID No. 19)D1 09+ GTTTTCCCAGTCACGACaagcacattttaccgatccag  5376  5396 38 210(SEQ ID No. 20) D1 09− AACAGCTATGACCATGgtcgtagtttctttctttctccttc  6299 6275 41 900 (SEQ ID No. 21) D1 10+GTTTTCCCAGTCACGACgcaatagacggggaatacag  6074  6093 37 182 (SEQ ID No. 22)D1 10− AACAGCTATGACCATGatgatggtggttttcagcag  6901  6882 36 809(SEQ ID No. 23) D1 11+ GTTTTCCCAGTCACGACgtgttgcttattccagagcc  6725  674437 138 (SEQ ID No. 24) D1 11− AACAGCTATGACCATGgctgtcttttccatttttctcc 7622  7601 38 877 (SEQ ID No. 25) D1 12+GTTTTCCCAGTCACGACactttgcacatcacagatcc  7354  7373 37 228 (SEQ ID No. 26)D1 12− AACAGCTATGACCATGttcgcactagcattcctcc  8192  8174 35 821(SEQ ID No. 27) D1 13+ GTTTTCCCAGTCACGACcacctgagaaatgtgacacc  7980  799937 175 (SEQ ID No. 28) D1 13− AACAGCTATGACCATGtttccttgtttatgaagctccc 8907  8886 38 907 (SEQ ID No. 29) D1 14+GTTTTCCCAGTCACGACcaaaagcgaaacgaggcac  8661  8679 36 207 (SEQ ID No. 30)D1 14− AACAGCTATGACCATGgtttcaccacacagtcatctcc  9575  9554 38 894(SEQ ID No. 31) D1 15+ GTTTTCCCAGTCACGACagaccagcgaaaaatggaac  9314  933337 221 (SEQ ID No. 32) D1 15− AACAGCTATGACCATGtcccaatgagccttctcac 1019610178 35 865 (SEQ ID No. 33) D1 16+GTTTTCCCAGTCACGACgctaatgctatctgttcagcc  9896  9916 38 262(SEQ ID No. 34) D1 16− AACAGCTATGACCATGtgattcaacagcaccattcc 10726 1070736 (SEQ ID No. 35) D1 16i+ ccatggaagctgtacgc 10480 10496 17(SEQ ID No. 36) D1 16i− gagacagcaggatctctgg 10671 10652 19 812 −28(SEQ ID No. 37)

2.2 Methods

2.2.1 Viral RNA Purification

From previous experience, a minimal of 1000 DICC₅₀ is required to get apositive RT-PCR reaction in the next steps. This means that a mimimumvirus titer of 10⁴ DICC₅₀/mL is necessary. Virus genomic RNA waspurified using QIAamp viral RNA mini kit (Qiagen), according to themanufacturer's recommendations. Briefly, a volume of 140 μl from a crudeviral sample was incubated in the presence of the lysis solution, andloaded onto a kit column. After washing steps, the purified viral RNAwas eluted by 60 μl of sterile nuclease-free water containing 1 μl (40units) of RNAse inhibitor (RNAse Out, Sigma).

2.2.2 Reverse Transcription

Viral RNA was reverse transcribed into cDNA by a reverse transcriptase(reverse iT) from ABGene. Again, standard operating conditions wereapplied, using 10 μl of purified RNA, in a final reaction volume of 20μl. The reaction was initiated by hybridization of the minus strandprimers. One RT reaction per PCR was performed (Table 1). The cDNAsynthesis was obtained by 45 min incubation at 47° C.

2.2.3 PCR

All PCR were performed with Expand High Fidelity PCR system (Rochediagnostics), using all 16 pairs of primers (+) and (−) from Table 1.PCR conditions were the following ones:

RT   2 μl 10x buffer  2.5 μl dNTP mix (10 mM)   2 μl Primers  0.8 μleach H2O 16.4 μl Enzyme  0.5 μl PCR program Denaturation 94° C.  2 minDenaturation 94° C. 15 sec Hybridization 55° C. 30 sec 40 cyclesElongation 68° C.  1 min Elongation 68° C.  5 min

All PCR products were controlled by electrophoresis on agarose gel.

2.2.4 Sequencing

The major part of the sequence reactions has been outsourced to GenomeExpress. Genome extremities, ambiguities, some inter-PCR junctions, andregions not sequenced by Genome Express for technical reasons wereperformed in-house.

Sequencing at Genome Express: PCR products were shipped at +4° C., andsequencing results were received as informatic sequence files. Textfile, quality files and chromatograms are available for each individualsequence. After sequence alignment, all discrepancies were checked onthe chromatogram, and corrected if identified as sequence algorithmerrors.

In-house sequencing: Sequence reactions were performed on thermocyclerPTC-200 (MJ Research), with Sequitherm Excell II LC kit (Epicentre).Each PCR product was sequenced on both strands independently in a singlereaction. Reactions were loaded onto a sequence electrophoresis gel. Runand analysis of sequence were performed on the automated sequencer GeneReadiR 4200 (Li-Cor).

Sequence Reaction

DNA up to 200/250 ng Reaction buffer 7.2 μl Primers (1-2 pM) 1.5 μl eachEnzyme 1 μl H2O up to 20 μl PCR program Denaturation 92° C.  2 minDenaturation 92° C. 15 sec Hybridization 50° C. 30 sec 30 cyclesElongation 70° C.  1 min Elongation 70° C. 10 sec

Addition of 3 μl of denaturating/loading buffer.

Denaturation of samples 3 min at 95° C. and ice cooling just beforesamples loading.

Sequence Electrophoresis

Electrophoresis parameters Gel parameters Voltage 1500 V Gel hight 41 cmCurrent  35 mA Gel thickness 0.2 mm Power  40 W Temperature 45° C. Runtime 9H00 Scan speed 3

2.3 Results

All PCR fragments were sequenced from both ends using a common PCR addedtail, i.e. a specific motif which has been added at 5′ end of allprimers:

(SEQ ID No. 38) 5′ primers: M13SEQ-GTTTTCCCAGTCACGAC (SEQ ID No. 39) 3′primers:  M13REV-AACAGCTATGACCATG

M13-SEQ and -REV sequences correspond to universal M13 primers motifs(New England Biolabs references).

For final contig assembly, a quick analysis was performed in Vector NTi,in ContigExpress module (Informax). The LAV1 reference sequence wascompared with all individual sequencing results. In such conditions, allresults could be aligned at the right place on the complete genome, evenwhen some regions were still missing contig assembly, giving a quickvisualization of the overall genome alignment.

2.3.1 Complete VDV1 Sequence Assembly

The final sequence alignment was performed in Vector NTi, AlignX module(Informax). The classical multiple sequence alignment algorithm ClustalW(Thompson et al., 1994) was used by the software to build the globalalignment. All the sequence results were aligned together with the LAV1reference sequence, thus allowing for a better reconstruction of thegenome. Any discrepancy in the sequence with regard to the referencerequired a confirmation on another independent sequence reaction. Thecomplete sequence of VDV1 is shown in SEQ ID No.1.

Some ambiguities are often found in single sequences, especially nearsequence extremities. This is inherent to the somewhat poor quality ofthe reaction at both ends of any PCR fragment. Such poor qualitysequences were excluded from the alignment, until two other independentsequence reactions were available from other PCR products. Discrepancytowards the reference was not taken into account in the final alignmentwhen not confirmed in at least two independent other PCR sequencesmatching the consensus. Conversely, any discrepancy confirmed on twoindependent sequences was kept in the final sequence.

Table 4 summarizes the characteristics of each individual sequencereaction, indicating start, end and length. Overlaps between adjacentPCR are also indicated, as well as differences with regard to thereference sequence in the last column.

TABLE 4 Dengue VDV1 individual sequences characteristics Name Start EndSize Overlap Comments D1 01+    35   921 886   0 D1 01−   899    33 866209 D1 02+   712  1596 884 D1 02−  1569   713 856 196 D1 03+  1415  2277862 D1 03−  2253  1400 853 160 D1 04+  2133  3027 894 2719 G >A (NS1-100 s) D1 04−  3000  2117 883 212 2719 G > A (NS1-100 s) D1 05+ 2834  3681 847 D1 05−  3654  2815 839 247 D1 06+  3451  4332 881 D1 06− 4325  3434 891 243 D1 07+  4113  4987 874 D1 07−  4961  4089 872 245D1 08+  4742  5564 822 D1 08−  5583  4742 841 183 D1 09+  5400  5916 516D1 09−  6274  5758 516 197 5962 C > A (NS3-481 N > K); 2 sequencesD1 10+  6114  6868 754 D1 10−  6883  6077 786 135 D1 11+  6761  7504 743D1 11−  7597  6733 864 217 D1 12+  7381  8034 653 7947 A >G (NS5-125 K > R) D1 12−  8143  7380 763 141 7947 A > G (NS5-125 K > R)D1 13+  8003  9730 727 D1 13−  8857  8002 855 182 D1 14+  8687  9472 785D1 14−  9544  8675 869 200 D1 15+  9344 10170 826 D1 15− 10162  9399 763253 D1 16+  9917 10261  344 2 sequences D1 16− 10706 10394  312 D1 16i+10500 10706  206   0 D1 16i− 10649 10204  455

The two extremities of the genome could not be sequenced from PCRamplification, because cDNA synthesis and PCR DNA reaction requiredoligonucleotides complementary to the ends of the genome. During theamplification step, these oligonucleotides are incorporated into the PCRfragment. The sequence result is that of the synthetic oligonucleotide,and not that of the virus itself. PCR from both ends of the virus genomedid work properly, suggesting that the viral sequence was notsignificantly different from the oligonucleotide sequence (if it hadbeen the case, PCR amplification should have failed or at least shouldhave been of poor quality). The two extremities of the genome could notbe distinguished from all other PCR amplifications. So, in thereconstructed genome (SEQ ID No.1), both genome ends were considered asidentical to oligonucleotide sequences (and also identical to thereference). At 5′ end, the sequence is that of nucleotides 1 to 34. At3′ end, the sequence is that of nucleotides 10707 to 10735.

2.3.2 Sequence Comparison

Direct sequence comparison between VDV1 strain and LAV1 reference showsa series of 3 nucleotides differences. Table 5 gives the complete listof these positions.

TABLE 5 Sequence comparison between LAV1 and  VDV1 strains NucleotidesAmino Acids Nt AA Position LAV1 VDV1 Region Position LAV1 VDV1 Notes2719 G A NS1 100 G G Silent 5962 C A NS3 481 N K 7947 A G NS5 125 K R

Nucleotide change in position 2719 is silent at the amino acid level.The second difference in position 5962 triggers an amino acid change atNS3-481 (asparagine to lysine). Both are hydrophilic, but lysine ispositively charged, whereas asparagine is not.

The last difference is located in NS5 peptide, substituting lysine toarginine in position NS5-125. Such amino acid substitution is relativelyconservative from a chemical point of view, both arginine and lysineresidues being hydrophilic and positively charged.

TABLE 6 Search of discrepancies on other Dengue 1 strains NucleotideNumber of strains position on sharing the same VDV1 strain nucleotide2719 24/40  5962 6/40 7947 1/40

When performing sequence alignment between all available Genbankserotype 1 Dengue genomic sequences, it appears that most of theidentified differences are also present on other strains (see Table 6).One position is unique in the VDV1 strain (position 7947; NS5-125).

Thus, the full genomic sequence of a VDV1 strain of the dengue virus hasbeen established.

Three nucleotide differences have been detected with regard to theparent LAV1 genomic sequence. VDV1 vaccine strain is derived from LAV1,through virus “sanitization” and passage from dog to monkey cells.

Differences between LAV1 and VDV1 can have several origins. First,cloning steps can elect a viral subpopulation that is not 100% identicalto the major sequence previously detected in LAV1. Second, LAV1 has beenproduced on PDK cells, whereas VDV1 has been made on Vero cells. Suchpassage from dog to monkey cells potentially induces virus changes thatreflect adaptation to the new cell line. Third, as for all RNA viruses,the lower viral RNA polymerase fidelity triggers a higher genomicmutation rate than DNA polymerases do.

In term of sequences only 3 differences between LAV1 and VDV1 wereobserved, corresponding to only 2 amino acids substitutions. All 14nucleotide positions that have been linked to LAV1 viral attenuation areconserved in VDV1. Furthermore the sequences of master and bulk VDV1have been compared (Table 7).

TABLE 7Dengue 1 nucleotide differences between wild type 16007 strain and attenuatedLAV1/PDK13 and VDV1 strains 2695/ 2719/ 2782/ 5063/ 5962/ 6048/ 6806/7330/ 7947/ 9445/ 1323/ 1541-3/ 1545/ 1567/ 1608/ 2363/ NS1- NS1- NS1-NS3- NS3- NS3- NS4a- NS4b- NS5- NS5- Virus E-130 E-203 E-204 E-211 E-225E-477 92 100 121 182 481  510 144 168 125 624 DEN-1 T GaA G A C A T G CG C A A A A C 16007 Val Glu Arg Gln Ser Met Asp Gly Ala Glu Asn Tyr MetGln Lys Ser LAV1/ C AaG A G T G C G T A C T G G A T PDK13 Ala Lys LysGln Leu Val Asp Gly Ala Lys Asn Tyr Val Gln Lys Ser VDV1 C AaG A G T G CA T A A T G G G T Master Ala Lys Lys Gln Leu Val Asp Gly Ala Lys Lys TyrVal Gln Arg Ser VDV1 C AaG A G T G C A T A A T G G G T Bulk Ala Lys LysGln Leu Val Asp Gly Ala Lys Lys Tyr Val Gln Arg Ser

Complete VDV1 master seed sequence was aligned with the bulk sequence.No difference between the two sequences was observed, indicating geneticstability across passages.

VDV1 shows a remarkable genetic stability with regard to its LAV1parent.

Example 3 Characterization

The objective of these studies was to assess whether changes inattenuation markers occurred through passages.

The flow chart shown on FIG. 2 summarises the developed manufacturingprocess that gives rise to the Filled Product (monovalent), “ready touse” doses

Briefly, after 2 successive passages on Vero cells of the ViralPre-Master Seeds delivered by the Research department, the respectiveworking seeds were obtained. The final virus cultivations were alsoconducted by infection of a Vero cell suspension. The viruses producedare then harvested. DesoxyRiboNucleic Acid (DNA) was digested accordingto an enzymatic treatment. Impurities were removed by ultrafiltration.Infectious titers were enhanced by a concentration step. An aqueousbuffered solution comprising cryoprotective agents (pH=7.5) is added andthis 0.22-μm filtrated mixture is then diluted at the targeted dosewithin the same solution. The active substance is then filled into glassvials, freeze-dried, and stored before use.

3.1 Phenotypic Markers

The results are shown in Table 8. The validated tests performed for themaster seed and the bulk are:

Plaque size: the assay was performed in Vero cells at 37° C. after 7days of incubation. Sizing of the plaques was performed by Saisamv.5.2.0 (Microvision Instruments) dedicated software, after imagecapture with a video camera. Two populations (0.3 mm and 0.8 mm) weredetected in LAV1. The major population was the smallest. Afteradaptation to Vero cells and biological cloning, VDV1 plaque sizedistribution appears homogenous, with more than 98% of the populationshowing a single peak, centered to 0.8 mm in diameter. These plaques areclearly distinct from plaques obtained with DEN-1 16007 virus (see FIGS.4 and 5).

Temperature sensitivity: monovalent 1 exhibits clear restricted growthat 39° C. with respect to the non-temperature sensitive (Ts), wild-type(WT) D1-16007. This was demonstrated both by infectious titer assay andby viral RNA quantification. Master, bulk and passage 18 (10 passagesafter the bulk passage) of the monovalent 1 seed display 90% or more oftiter reduction at 39° C., compared to 37° C.

TABLE 8 Summary of DEN-1 viral phenotypes Neurovirulence Temperaturesensitivity in newborn Swiss (Percent titer reduction at 39°C.)_(Fold-reduction) Webster mice Virus Score Day 3 Day 4 Day 5 Day 6Mortality_(n)* AST (S.D.) D1-16007 − 62.1_(2.6) 59.3_(2.5 ) 56.3_(2.3 )(−28.5_(−1.4))  6.25%₁₆ 19.0 (0.0) D1-PDK13 + 87.1_(7.8) 91.3_(11.5)95.6_(22.2) 96.5_(28.6) 0.00%₁₆ n.a. VDV1 MS +  97.2_(35.7) 97.7_(43.5)98.8_(83.3)  99.5_(200.0) 0.00%₁₆ n.a. *_(n)number of animals

3.2 Genotypic Markers

VDV1 vaccine strain can be distinguished from parental strains at thegenomic level. Attenuation-specific loci have been identified. Theseloci are conserved in master and bulk seeds.

Example 4 Immunogenicity, Viremia, and Toxicology in Monkeys

The most solid and numerous data that can be obtained in monkeys concernimmunogenicity and viremia. Viremia, in particular, has been identifiedas one of the factors associated with virulence and disease severity inhumans, and then constitutes an important parameter to consider.Obviously, immunogenicity is a key parameter when testing vaccines.

Inventors have established minimal/maximal values for viremia andimmunogenicity.

TABLE 9 Minimal requirements for responses induced by Dengue vaccinecandidates in monkeys, as measured in Vero or LLC-MK2 cells by plaqueassay (these cells being considered equivalent in such an assay) Viremiamean duration Viremia mean peak titer Mean neutralizing titer (days)(log 10 pfu) Day 30 (all serotypes being (all serotypes being (for eachserotype) considered) considered) PRNT 50 ≦3 days ≦1.5-2 ≧80 pfu: plaqueforming unit PRNT 50: Plaque Reduction Neutralization Titer 50 (titrecorresponding to a reduction of 50% of plaque number)

4.1 Material and Methods

4.1.1 Monkey Experiments

Monkey experiments were carried out according to European guidelinesregarding animal experiments.

Immunizations were performed on cynomolgus monkeys (Macaca fascicularis)originating from Mauritius (CRP Le Vallon). Monkeys were quarantined for6 weeks in the animal facility of Sanofi Pasteur before immunization.

Monkeys were immunized by subcutaneous (SC) route in the arm withvaccines in a volume of 0.5 ml (see each respective section). Afterlight anesthesia with ketamine (Imalgene, Merial), blood was collectedby puncture of the inguinal or saphene veins. At days 0 and 28, 5 ml ofblood were sampled for evaluating antibody responses while between days2 and 10 only 1 ml of blood was sampled for evaluating viremia. Bloodwas collected on ice and kept on ice until serum separation. To do so,blood was centrifuged for 20 minutes at 4° C., and serum collected andstored at −80° C. until testing in Rich Kinney's laboratory. Shipment toUSA was performed in dry ice.

4.1.2 Viremia and Neutralizing Antibody Responses (Plaque ReductionNeutralization Test, PRNT)

All analyses were performed in the laboratory of R. Kinney in CDC, FortCollins, USA. Serum samples were shipped and stored at −80° C. until thetime of testing. At the time of first thawing, the samples were testedfor viremia, and a 1:5 dilution of the serum was made. The 1:5 serumdilutions were inactivated for 30 min at 56° C. before testing forneutralizing antibodies.

4.1.2.1 Viremia

0.125 ml of serum was added to 0.125 ml of diluent (RPMI medium) in thefirst well of 96-well plate and serial 10-fold dilution series weredone, transferring 0.025 ml into 0.225 ml of diluent for each dilution.0.2 ml of 10^(0.3)-10^(5.3) dilution series was plated in 6-well plateof Vero cells (virus was adsorbed at 37° C. for 1.5 hour, overlayed with4 ml of agarose lacking neutral red, overlayed 6-7 days later with 2 mlof agarose containing neutral red, and plaques counted). The limit ofvirus detection was =10 PFU/ml. For controls stock DEN-16007 PDK-13(LAV1) vaccine was plated.

4.1.2.2 PRNT (Plaque Reduction Neutralization Test)

Neutralizing antibodies were quantified as described in Huang et al.(2000). Briefly, 0.2 ml of heat-inactivated, 1:5 dilution of serum wasadded to the first well of 96-well plate and serial 2-fold dilutionseries were made, transferring 0.1 ml into 0.1 ml of diluent (RPMImedium) for each dilution. This resulted in a 1:10-1:320 serum dilutionseries. 0.1 ml of DEN virus (60-160 PFU; parental DEN1 16007 virus) wasadded to each serum dilution well for a total of 0.2 ml of serum-virusmixture. 96-well plates were incubated overnight at 4° C. 0.1 ml ofserum-virus mixtures (containing 30-80 PFU of input virus) were platedin 6-well Vero plates (as indicated above in the Viremia section) andplaques were counted after staining with neutral red. Multiple backtitrations of the input viruses at 2-fold, 1-fold, and 0.5-fold testconcentrations provided direct experimental determination of the inputPFU, which was the basis for determining 50% (PRNT₅₀) and 70% (PRNT₇₀)endpoint antibody titers. A negative serum result should have aneutralizing antibody titer of <1:10. Sera showing neutralization titersof 320 were retested at dilutions 1:80-1:2560 for determination ofendpoint titer.

4.2 Evaluation of VDV Candidates

4.2.1 VDV 1/Pre-Master

Purification/selection of D1 candidate has been conducted as describedin example 1. The selected clones (based on phenotypic markers andsequence) have been tested in sanofi pasteur as described in Materialand Methods (Marcy I'Etoile animal facility, I15) on male cynomolgusmacaques (Macaca fascicularis, mean weight 3.1 kg) originating from CRPLe ValIon, Mauritius.

After immunization on D0, viremia was followed from D2 to D10, andimmunogenicity measured at D0 and D28. All viruses and vaccines, when inliquid form, were kept at −70° C.

LAV1: titre: 10^(3,9) DICC₅₀/ml; lyophilized, resuspended in 0.5 ml ofPBS (containing Ca²⁺ and Mg²⁺; CaCl₂.2H₂O 0.133 g/l; MgCl₂.6H₂O, 0.1g/l) and administered in totality.

Premaster VDV1 DEN1-TV111: Titre: 10^(5,9) DICC₅₀ /ml; liquid, dilutedat 10^(5,3) pfu/ml in PBS (containing Ca²⁺ and Mg²⁺; CaCl₂.2H₂O 0,133g/l; MgCl₂.6H₂0, 0.1 g/l); 0.5 ml administered.

Injection was done by SC route in the arm with a 23G1 needle, at a 10⁵DICC₅₀ dose for VDV1.

The results are as presented in Table 10. Titrations at day 28 werecarried out in triplicate (PRNT ₇₀) or in duplicate (PRNT₅₀).

TABLE 10 VDV1 PreMaster immunogenicity AvP monovalent VDV1 (Exp A) DENMonkey study (F. Mi.DEN003.Si): PRNT and Viremia Results NeutralizingAntibody Titer Viremia (PFU/ml in Vero cells) Day (−15) Day 28 Day DayDay Day Day Day Day Day Day Day Serum Group PRNT₇₀ PRNT₅₀ PRNT₇₀ PRNT₅₀−15 2 3 4 5 6 7 8 9 10 AD 333 LAV DEN-1 <10/<10 <10 320/160/160 160/3200 0 0 0 0 0 0 0 0 0 AC 763 <10/<10 <10 640/640/640 1280/1280 0 0 0 0 0 00 0 50 150 AD 209 <10/<10 <10 320/160/160 160/320 0 0 0 0 0 0 0 0 0 0 AC755 <10/<10 <10 160/80/160 160/160 0 0 0 0 0 0 50 0 0 0 AC 775 VDV DEN-1<10/<10 <10 160/80/80 160/160 0 0 0 0 0 0 0 0 0 0 AC 881 TV111 <10/<10<10 20/10/10 20/20 0 0 0 0 0 0 0 0 0 0 AD 145 <10/<10 <10 320/80/80160/80  0 0 0 0 0 0 0 0 0 0 AD 113 <10/<10 <10 20/10/10 20/20 0 0 0 0 00 0 0 0 0 Virus Exp#1 Exp#2 Exp#3 DEN-1 112 PFU 45 PFU 101 PFU

Briefly, responses were rather homogeneous within each group, and someclear tendencies could be identified for each construct. No dramaticdifferences were found between VDV1 and LAV1: low and late viremia wasobserved in some LAV1 monkeys. VDV1 looked satisfactory, and inparticular presented no viremia.

4.2.2 VDV 1 Bulk

As immunogenicity of the vaccines had been tested at the Premasterstage, a further experiment was designed to test each monovalent at theBulk stage.

Male Macaca fascicularis monkeys were used as before, originating fromC.R.P. Le Vallon, Ile Maurice (24 monkeys, mean weight 3.4 kg).

VDV1; Batch: Titre: 8.37 log10 DICC₅₀ /ml

Placebo: PBS with Ca²⁺ and Mg²⁺

Vaccines were diluted at 10^(5.3) DICC₅₀/ml in PBS (containing Ca²⁺ andMg²⁺; CaCl₂.2H₂O 0.133 g/l; MgCl₂.6H₂O, 0.1 g/l); 0.5 ml administered bySC route in the arm with a 23G1 needle, corresponding to a dose of 10⁵DICC₅₀.

Viremia and immunogenicity have been measured as usual in CDC by RKinney. The results are shown in Table 11.

VDV1 monovalent vaccine induced a significant immune response whileviremia was absent. Thus, this monovalent VDV1 fulfilled the successcriteria initially defined in monkeys.

TABLE 11 VDV Bulk VDV1 immunogenicity and viremia Monkey study(F.MI.DEN004.Mk): Monovalent and Tetravalent VDV1 Neutralizing AntibodyTiter Viremia (PFU/ml in Vero cells) Day (−14) Day 28 Day Day Day DayDay Day Day Day Day Monkey Group PRNT₅₀ PRNT₇₀ PRNT₅₀ PRNT₅₀ −14 2 3 4 56 7 8 9 AE 484 VDV DEN-1 — — 14 5 0 0 0 0 0 0 0 0 0 AE 627 — — 8122 45580 0 0 0 5 0 0 0 0 AF 115 — — 359 202 0 0 0 0 0 0 0 0 0 AF 227 — — 557367 0 5 0 5 0 0 0 0 0 Geo Homologous — — 388 203 Mean response AE 538Placebo —/—/—/— —/—/—/— 2.5/—/2/2 —/—/—/— 0 0 0 0 0 0 0 0 0 AE 548—/—/—/— —/—/—/— —/—/1/2 —/—/—/— 0 0 0 0 0 0 0 0 0 AE 556 —/—/1.5/2—/—/—/— 1/—/—/— —/—/—/— 0 0 0 0 0 0 0 0 0 AE 572 —/—/1.5/5 —/—/1.5/25/—/—/2 —/—/—/— 0 0 0 0 0 0 0 0 0 Geo Response —/—/1.2/3 —/—/1/12/—/1.2/1.6 —/—/—/— Mean against the four serotypes D 1/D 2/ D 1/D 2/ D1/D 2/ D 1/D 2/ D 3/D 4 D 3/D 4 D 3/D 4 D 3/D 4

4.3 Neurovirulence Tests in Monkeys

For each virus type, 10 cynomolgus monkeys from Mauritius wereinoculated with VDV1 master seed by the intracerebral route (10^(7.23)CCID₅₀/mL in the thalamus of each hemisphere). At the end of the test,the monkeys were sacrificed and perfused with formaline solution. Tissuesamples were taken from the brain of each monkey (medulla oblongata,pons and cerebellum, midbrain, thalamus including the left and the rightparts, the left and the right of the cerebral cortex). Sections were cutat a thickness of 8 μm and stained by eosin and gallocyanin.

No histopathological signs of pathogenicity were observed in the monkeybrains injected with VDV1 seeds.

Example 5 Safety of Monovalent VDV1 in Healthy, Flavivirusnaive AdultsAged 18 to 40 Years

The aim of this phase 1 trial is to document the safety, viremia, andimmunogenicity profiles of monovalent VDV1 at a virus concentration of10⁴ CCID₅₀ compared to Stamaril® (used as control group) inflavivirus-naive adults. Single injections are given, with follow-up at6 and 12 months. For safety precaution, sequential inclusions areperformed in the study.

Enrollment and vaccinations are therefore staggered; a 1st cohort (n=4per group, total n=12) have been vaccinated. The safety data collectedup to Day 28 have been reviewed by an Independent Data MonitoringCommittee (IDMC) and by the Royal Adelaide Hospital InvestigationalDrugs Subcommittee (IDSC) before deciding to proceed with thevaccination of the remaining subjects (n=8per group, total n=16). Aschematic representation of the trial design is provided in FIG. 6.

After administration of the vaccine the patient are regularly submittedto various clinical examination and testing. A summary of this follow upis given in table 12 below.

The enrolled population consists of adults aged 18 to 40 years (i.e. theday of the 18th birthday to the day before the 41st birthday) on day ofinclusion who are flaviviruses-naive [persons presenting vaccinationagainst flavivirus diseases (e.g. yellow fever, Japanese encephalitis,dengue fever); or history of flavivirus infection (confirmed eitherclinically, serologically or microbiologically) or previous residence inor travel to areas with high dengue infection endemicity (whatever theduration), or residence in or travel to North Queensland for 2 weeks ormore) were excluded]

TABLE 12 Flow chart for follow up Visit Number V 01 V 02 V 03 V 04 V 05V 06 V 07 V 08 V 09 V 10 V 11 V 12 Trial timelines 

D 0 D 2 D 4 D 6 D 8 D 10 D 12 D 14 D 16 D 28 D 180 D 365 Time Windows ±1d ±1 d ±4 d ±15 d ±30 d Clinical Examination ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Vital signs (BP, pulse ✓ rate) Oral temperature ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Blood Sampling: Serology HBV/HCV/HIV ✓ ✓ ✓ ✓ ✓ ✓ Biological Safety ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ Viremia ✓ ✓ ✓ ✓ ✓ Immunogenicity ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Cytokines in serum ✓ ✓ PBMCs for T cell ✓ ✓ (subset) immediatesurveillance ✓ Local & systemic events ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ V:visit - D: day

 Time intervals between visits will be calculated from the date of studyvaccination which might differ from the date of visit (e.g. in case atemporary exclusion criterion is met). V 06 and V 07 must be done withat least 1-day interval.

The products tested are:

The vaccine evaluated is a lyophilised product in a vial that isreconstituted extemporaneously with the diluent provided separately:

Active ingredient: 4±0.5 log₁₀ CCID₅₀ of either monovalent Vero denguevirus serotype 1 (VDV1) per 0.5 mL dose;

Diluent: Sterile NaCl 4% solution for vaccine reconstitution.

The reconstituted vaccine, i.e 0.5 mL of NaCl 4%0 solution of monovalentVDV1, should be used immediately or be maintained until use +2° C. and+8° C.

The 0.5 mL vaccine dose is administered subcutaneously in the deltoidregion.

The control vaccine Stamaril®, is a yellow fever vaccine produced byAventis Pasteur. Stamaril® is presented as a lyophilised,avian-leukosis-free, stabilised product to be reconstituted with adiluent immediately before use. (Active ingredient: Live attenuatedyellow fever virus (17D strain): ≧1,000 mouse Lethal Dose 50%(LD₅₀)/Diluent: Sterile NaCl 4% solution).

The control vaccine is administered subcutaneously in the deltoidregion.

The preliminary results of the trial are reported in Table 13 below.

TABLE 13 preliminary safety data Day Day Day Day Day Day Day Day Day DayDay Day Day Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 LOCAL SOLICITED Pain 1Erythema Induration 1 Edema LOCAL UNSOLICITED Bruise 1 1 Pruritis OTHERSOLICITED Temp ≧ 37.5 C. 1 1 Rigors 1 1 1 Malaise 2 1 1 1 1 1 1 1 2 1 2Asthenia 1 2 1 1 2 1 1 2 1 1 Anorexia 1 1 1 1 1 1 Nausea 1 2 1 1 1 1 1 12 Vomiting 1 1 1 1 1 1 Stomach Pain 1 1 2 2 1 1 1 1 1 1 1 Headache 2 1 22 1 1 1 2 1 1 1 1 Myalgia 1 1 1 1 1 2 Arthralgia 1 1 1 1 1 1 1 Avoidanceof 1 1 1 light Conjunctivitis Eye Pain 1 1 RASH Macular 1 (1%) Papular 1(1%) Maculo- 1 1 papular (90%) (90%) OTHER UNSOLICITED Decreased 2 1 WCCNeutropenia 2 1 Increased 1 1 aPPT Elevated CK 1 Odd dreams 1 Low abdopain 1 1 1 (kidneys/liver) Diarrhoea 1 1 1 1 1 1 1 1 1 Sore throat 1 1Cough 1 Early 1 menstruation Tiredness 1

Table 13 shows that biological abnormalities (WCC reductions, plateletcount reductions) have all been mild. The symptoms have been mainlymalaise, nausea, diarrhoea and occasional vomiting. They have been ofmoderate severity. One significant rash—typical “viral” maculopapularrash, onset day 12, 90% coverage.

The safety data of the second cohort are also satisfactory with nobiological abnormality recorded. All subjects have antibody response 28days after vaccination against dengue 1 (titer between 1315 and 13150).

REFERENCES

The following references are incorporated herein by reference as if setforth in their entirety herein:

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1. An isolated polyprotein encoded by SEQ ID No.1, or fragments thereofthat comprise at least a lysine at position 481 of NS3 protein and/or anarginine at position 125 of NS5 protein.
 2. The isolated polyprotein orfragments thereof according to claim 1, wherein the polyprotein is thepolyprotein of sequence SEQ ID No.41.
 3. A fragment of the polyproteinaccording to claim 1 which is at least 20, amino acid long.
 4. Afragment of the polyprotein according to claim 1 which comprises NS3protein and/or NS5 protein.